Method to grow Actinobacillus pleuropneumoniae biofilm on a biotic surface

Abstract

Background: Actinobacillus pleuropneumoniae is a Gram-negative bacterium and a member of the Pasteurellaceae family. This bacterium is the causative agent of porcine pleuropneumonia, which is a highly contagious respiratory disease causing important economical losses to the worldwide pig industry. It has been shown that A. pleuropneumoniae can form biofilms on abiotic surfaces (plastic and glass). Although in vitro models are extremely useful to gain information on biofilm formation, these models may not be representative of the conditions found at the mucosal surface of the host, which is the natural niche of A. pleuropneumoniae.

Results: In this paper, we describe a method to grow A. pleuropneumoniae biofilms on the SJPL cell line, which represents a biotic surface. A non-hemolytic, non-cytotoxic mutant of A. pleuropneumoniae was used in our assays and this allowed the SJPL cell monolayers to be exposed to A. pleuropneumoniae for longer periods. This resulted in the formation of biofilms on the cell monolayer after incubations of 24 and 48 h. The biofilms can be stained with fluorescent probes, such as a lectin against the polymer of N-acetyl-D-glucosamine present in the biofilm matrix, and easily observed by confocal laser scanning microscopy.

Conclusions: This is the first protocol that describes the formation of an A. pleuropneumoniae biofilm on a biotic surface. The advantage of this protocol is that it can be used to study biofilm formation in a context of host-pathogen interactions. The protocol could also be adapted to evaluate biofilm inhibitors or the efficacy of antibiotics in the presence of biofilms.

Figure 1

Effect of incubation time on the biofilm dispersion of A. pleuropneumiae strain S4074 and MBHPP147 in 96-well polystyrene microtiter plates.

Figure 2

Confocal laser scanning microscopy images of 6-hour biofilms of A. pleuropneumoniae strain MBHPP147 stained with WGA-Oregon green 488. Biofilms were formed in 6-well and 96-well polystyrene plates.

Figure 3

Confocal laser scanning microscopy images of biofilms of A. pleuropneumoniae strain MBHPP147 grown on SJPL cells and stained with WGA-Oregon green 488. Uninfected SJPL cell control (48 hours post mock infection) (A); SJPL cells incubated with A. pleuropneumoniae strain MBHPP147 for 3 hours (B), 6 hours (C), 24 hours (D), 48 hours (E) or 48 hours and treated with Dispersin B (F).

Figure 4

Number of colony forming units (CFU) attached to SJPL cells. SJPL cells were infected with A. pleuropneumoniae strain MBHPP147 and attached cells were recovered after 3, 6, 24, and 48 hours of incubation and after a Dispersin B treatment (48 h + Dispersin B).

Figure 5

Confocal laser scanning microscopy images of a 24 hour biofilm of A. pleuropneumoniae strain MBHPP147 expressing GFP grown on SJPL cells and stained with Phalloidin-AlexaFluor 594 and WGA-AlexaFluor 633.